Acyltransferase for synthesis of polyester intermediates and use thereof

Abstract

The application discloses an acyltransferase for synthesizing polyester intermediates and application thereof. The acyltransferase is specifically a wild-type enzyme or a mutant acyltransferase variant with catalytic acyltransferase activity, which is obtained by substituting at least one of amino acid residues at positions 209, 166 and 119 in the amino acid sequence of the wild-type enzyme. The acyltransferase variant is specifically a protein with improved acyltransferase activity. Both the wild-type enzyme and the acyltransferase variant provided by the application have acyltransferase activity. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to an acyltransferase for synthesizing polyester intermediates and its applications. Background Technology

[0002] Studies have found that some hydrolases (such as MsAcT from Mycobacterium smegmatis, EstCE1 from family VIII carboxylesterases, and Est8 from metagenomics) can catalyze the synthesis of esters and amides in water in addition to their hydrolytic function. These enzymes are called hybrid hydrolases / acyltransferases. Compared with traditional chemical synthesis and lipase synthesis, the synthetic reactions catalyzed by hybrid hydrolases / acyltransferases do not require the participation of expensive and toxic organic solvents and can be used for multi-enzyme-driven biocatalytic cascade reactions, showing broad application prospects. However, currently only a small number of hydrolases have had their acyltransferase functions explored, and their ester synthesis reactions catalyzed in water are usually kinetically controlled, generally suffering from low product yields and severe hydrolytic side reactions. There is an urgent need to solve these problems through enzyme molecule modification strategies, i.e., to obtain hydrolases with high acyltransferase activity.

[0003] PETase, a PET hydrolase derived from Ideonella sakaiensis 201-F6, can hydrolyze the recalcitrant plastic polyethylene terephthalate (PET) into monomers terephthalic acid (TPA) and ethylene glycol (EG) at 20-40°C. PETase is widely studied and used as a plastic hydrolase. Summary of the Invention

[0004] The purpose of this invention is to provide an acyltransferase for synthesizing polyester intermediates. Specifically, the acyltransferase is a hydrolase PETase (wild type) with an amino acid sequence as shown in SEQ ID NO: 1, or a variant thereof obtained by mutation, which has the activity of catalyzing acyl transfer reactions.

[0005] This invention first protects proteins, which can be any of C1-C4:

[0006] C1) Replace at least one of the three amino acid residues at positions 209, 166, and 119 in the amino acid sequence of the hydrolase PETase to obtain a protein with acyltransferase activity.

[0007] C2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in C1);

[0008] C3) A protein with acyltransferase activity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in C1) or C2).

[0009] C4) is a protein that has 80% or more homology with the amino acid sequence of the protein shown in C1) or C2) and has acyltransferase activity.

[0010] The amino acid sequence of the hydrolase PETase is shown in SEQ ID NO: 1.

[0011] The protein in question is an acyltransferase variant derived from a mutation of the hydrolase PETase.

[0012] In the aforementioned protein, the A residue at position 209 is replaced with a T residue, V residue, N residue, G residue, I residue, C residue, F residue, Q residue, R residue, M residue, D residue, P residue, L residue, K residue, H residue, W residue, or Y residue. The S residue at position 166 is replaced with a T residue, V residue, C residue, E residue, M residue, or G residue. The Q residue at position 119 is replaced with an R residue, C residue, W residue, P residue, F residue, I residue, or G residue.

[0013] In the above text, positions 209, 166, and 119 in the amino acid sequence of the hydrolase PETase refer to positions 209, 166, and 119 from the N-terminus of SEQ ID NO: 1, respectively.

[0014] The acyltransferase activity of any of the proteins described above is higher than that of the hydrolase PETase.

[0015] The protein described above can be any one of the 37 acyltransferase mutants in Table 1 of the examples (i.e., acyltransferase mutants other than Q119L and Q119S).

[0016] Nucleic acid molecules encoding any of the proteins described above are also within the scope of protection of this invention.

[0017] Expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the aforementioned nucleic acid molecules are also within the scope of protection of this invention.

[0018] The recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or cloning vector. Specifically, the expression vector may be the pET-22b(+) vector mentioned in the examples.

[0019] The recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant vector into the starting microorganism.

[0020] The originating microorganism may be Escherichia coli.

[0021] The specific Escherichia coli mentioned may be Escherichia coli BL21 Gold(DE3).

[0022] The application of any of the aforementioned proteins, the aforementioned hydrolase PETase, any of the aforementioned nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the aforementioned nucleic acid molecules in the preparation of acyltransferases is also within the scope of protection of this invention.

[0023] The use of any of the proteins, the hydrolase PETase, the nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms described above as acyltransferases is also within the scope of protection of this invention.

[0024] In the above applications, the acyltransferase activity of any of the proteins described above is higher than that of the hydrolase PETase; the amino acid sequence of the hydrolase PETase is shown in SEQ ID NO: 1.

[0025] The application of any of the proteins, nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms described above in PET hydrolysis is also within the scope of protection of this invention.

[0026] The application of any of the aforementioned proteins, the aforementioned hydrolase PETase, any of the aforementioned nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms in catalyzing the acyl transfer reaction of BHET is also within the scope of protection of this invention.

[0027] The application of any of the aforementioned proteins, the aforementioned hydrolase PETase, any of the aforementioned nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the aforementioned nucleic acid molecules in catalyzing acyl transfer reactions of aromatic, furan, or aliphatic polymer precursors is also within the scope of protection of this invention.

[0028] The aromatic polymer precursor may be bis(4-hydroxybutyl)terephthalate, 2-hydroxyethyl benzoate, 4-hydroxybutyl benzoate, or 6-hydroxyhexyl benzoate.

[0029] The furan polymer precursor may be bis(2-hydroxyethyl)furan-2,5-dicarboxylate or bis(4-hydroxybutyl)furan-2,5-dicarboxylate.

[0030] The aliphatic polymer precursor may be 2-hydroxyethyl 3-phenylpropanoate, 4-hydroxybutyl 3-phenylpropanoate, or 6-hydroxyhexyl 3-phenylpropanoate.

[0031] bis(4-hydroxybutyl)terephthalate, bis(2-hydroxyethyl)furan-2,5-dicarboxylate, bis(4-hydroxybutyl)furan-2,5-dicarboxylate, 2-hydroxyethyl 3-phenylpropanoate, 4-hydroxybutyl 3-phenylpropanoate, 6-hydroxyhexyl 3-phenylpropanoate, 2-hydroxyethyl The structural formulas of benzoate, 4-hydroxybutyl benzoate and 6-hydroxyhexyl benzoate are detailed in Table 5 in the Examples.

[0032] The application of any of the aforementioned proteins, the aforementioned hydrolase PETase, any of the aforementioned nucleic acid molecules, or expression cassettes containing any of the aforementioned nucleic acid molecules, recombinant vectors, or recombinant microorganisms in the catalytic synthesis of esters and amides is also within the scope of protection of this invention.

[0033] Given that the hydrolase PETase, with an amino acid sequence as shown in SEQ ID NO: 1, possesses weak acyltransferase activity, the inventors of this invention have conducted extensive experiments to explore and modify the hydrolase PETase. Specifically, they have performed point mutations on the existing hydrolase PETase with an amino acid sequence as shown in SEQ ID NO: 1, preparing a series of PETase mutants with significantly enhanced acyltransferase activity, thereby achieving efficient ester synthesis in the aqueous phase. This invention has significant application value. Attached Figure Description

[0034] Figure 1 The purification results are for the hydrolase PETase (i.e., PETase WT), the acyltransferase mutant A209T / S166V (i.e., A209T / S166V), the acyltransferase mutant A209T (i.e., A209T), and the acyltransferase mutant S166V (i.e., S166V).

[0035] Figure 2 For BHET-MHET 1H-NMR spectrum.

[0036] Figure 3 For BHET-MHET 13 C-NMR spectrum.

[0037] Figure 4 For substrate 1 in Example 8 1 H-NMR spectrum.

[0038] Figure 5 For product 1p in Example 8 1 H-NMR spectrum.

[0039] Figure 6 For substrate 2 in Example 8 1 H-NMR spectrum.

[0040] Figure 7 For product 2p in Example 8 1 H-NMR spectrum.

[0041] Figure 8 For substrate 3 in Example 8 1 H-NMR spectrum.

[0042] Figure 9 For product 3p in Example 8 1 H-NMR spectrum.

[0043] Figure 10 For substrate 4 in Example 8 1 H-NMR spectrum.

[0044] Figure 11 For product 4p in Example 8 1 H-NMR spectrum.

[0045] Figure 12 For substrate 5 in Example 8 1 H-NMR spectrum.

[0046] Figure 13 For product 5p in Example 8 1 H-NMR spectrum.

[0047] Figure 14 For substrate 6 in Example 8 1 H-NMR spectrum.

[0048] Figure 15 For product 6p in Example 8 1 H-NMR spectrum.

[0049] Figure 16 For substrate 7 in Example 8 1 H-NMR spectrum.

[0050] Figure 17 For product 7p in Example 8 1 H-NMR spectrum.

[0051] Figure 18 For substrate 8 in Example 8 1 H-NMR spectrum.

[0052] Figure 19 For product 8p in Example 8 1 H-NMR spectrum.

[0053] Figure 20 For substrate 9 in Example 8 1 H-NMR spectrum.

[0054] Figure 21 For product 9p in Example 8 1 H-NMR spectrum. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1: Preparation of hydrolase PETase (abbreviated as PETase or PETase WT) and PETase mutants (also known as acyltransferase variants or acyltransferase mutants).

[0058] I. Construction of Recombinant Plasmids

[0059] 1. Replace the small DNA fragment between the recognition sites of the restriction endonucleases EcoRI and XhoI in the pET-22b(+) vector with the PETase hydrolase gene (Genebank number GAP38373.1) from the Ideonella sakaiensis 201-F6 strain to obtain the recombinant plasmid pET22b-PETase.

[0060] The recombinant plasmid pET22b-PETase expresses the PETase shown in SEQ ID No: 1. PETase exhibits weak acyltransferase activity.

[0061] 2. Using primers F: 5'-GGCCATGGATATCGGAATTAATTCGGATCCGAATTCG-3' and R: 5'-TCAGTGGTGGTGGTGGTGGTGCTCGAG-3', the nucleotide sequence of the hydrolase PETase gene from the Ideonella sakaiensis 201-F6 strain was randomly mutated by error-prone PCR to obtain several mutant nucleotide sequences.

[0062] 3. Replace the small DNA fragment between the recognition sites of the restriction endonucleases EcoRI and XhoI in the pET-22b(+) vector with several mutant nucleotide sequences obtained in step 2 to obtain the corresponding recombinant plasmid pET22b-PETase mutant.

[0063] II. Obtaining Acyltransferase Mutants

[0064] Each recombinant plasmid constructed in step one was subjected to the following experiments:

[0065] 1. Transform the recombinant plasmid into Escherichia coli BL21 Gold(DE3) to obtain the recombinant bacteria.

[0066] 2. Select a single clone of recombinant bacteria and inoculate it into 150 μL of LB liquid medium (containing ampicillin). Incubate overnight at 37°C and 800 rpm in a 96-well plate shaking incubator to obtain the culture solution.

[0067] 3. Inoculate the culture medium obtained in step 2 into 150 μL of LB liquid medium (containing ampicillin), incubate at 37℃ for 3 h, add IPTG to the final concentration of 0.1 mM, incubate at 20℃ and 800 rpm for 24 h with shaking, centrifuge at 4℃ and 4000 rpm, and collect the supernatant.

[0068] 4. Preparation of the screening system. Screening was carried out in a 96-well plate. The screening system consisted of 200 μL of pH 8.0, 100 mM phosphate buffer, the supernatant collected in step 3, and 15 mM diethyl terephthalate (BHET) solution.

[0069] 5. Take the screening system prepared in step 4 and react it at 30℃ for 24 hours; use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD at different time points. 600nm .

[0070] Compared with recombinant bacteria transformed by the recombinant plasmid pET22b-PETase, OD 600nm The acyltransferase activity of the mutant expressed in recombinant bacteria transformed with the significantly increased recombinant plasmid pET22b-PETase was significantly enhanced. These OD... 600nmGene sequencing was performed on the significantly increased recombinant bacteria, and the mutation sites were determined based on the sequencing results to obtain the amino acid sequence of the PETase mutant with enhanced acyltransferase activity.

[0071] The names of the acyltransferase mutants obtained through screening are shown in column 1 of Table 1. The different amino acid residue substitutions of the acyltransferase mutants compared with the amino acid sequence of PETase (the mutation site corresponds to the sequence shown in SEQ ID No: 1) are shown in column 2 of Table 1.

[0072] Table 1

[0073]

[0074]

[0075] Preparation of crude enzymes from acyltransferase mutants screened in Example 2 and Example 1

[0076] The inoculation needle picked up recombinant bacteria transformed from the recombinant plasmid pET22b-PETase and recombinant bacteria transformed from the recombinant plasmid pET22b-PETase mutant obtained in Example 1, and inoculated them into 3 mL of LB liquid medium. The cultures were incubated overnight at 37°C and 220 rpm. Then, 1% (V / V) inoculation was carried out into 15 mL of LB liquid medium and incubated at 37°C and 220 rpm until OD (out of control) was reached. 600nm The concentration was increased to approximately 0.8. Then, IPTG was added to a final concentration of 0.1 mM and the mixture was induced to express for 24 h at 20 °C and 220 rpm. After centrifugation at 4 °C and 4000 rpm for 30 min, the supernatant was collected.

[0077] The supernatant is the corresponding crude enzyme.

[0078] Example 3: Preparation of purified enzymes of the acyltransferase mutants screened in Example 1

[0079] Purify the supernatant collected in Example 2 according to the instructions for the nickel column (GE Healthcare product, catalog number 17-5248-02), and store the final eluted target protein in 100mM pH 8.0 phosphate buffer after removing imidazole.

[0080] The target protein obtained by purifying the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase through the above purification steps is the pure enzyme PETase.

[0081] The target protein obtained by the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase mutant obtained in Example 1, after purification through the above steps, is the pure enzyme of the acyltransferase mutant.

[0082] Partial purification results are shown below Figure 1(1 is the purified PETase enzyme, 2 is the purified acyltransferase mutant A209T / S166V enzyme, 3 is the acyltransferase mutant A209T, and 4 is the acyltransferase mutant S166V). The results showed that both the purified PETase enzyme and the purified acyltransferase mutant enzyme had virtually no impurity bands, indicating good protein purification efficiency.

[0083] Example 4: Enzymatic preparation and identification of the acyl transfer product O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl) terephthalate (BHET-MHET)

[0084] 1. Add 50 mL of 100 mM, pH 8.0 phosphate buffer and 0.5 g of BHET powder to a 250 mL Erlenmeyer flask, followed by 50 μL of purified acyltransferase mutant A209T / S166V. Incubate the reaction at 30 °C and 220 rpm with shaking. After the reaction is complete, centrifuge the reaction solution (4 °C, 12000 rpm, 10 min), collect the precipitate, wash 3–5 times with ddH2O, completely dissolve the precipitate with acetonitrile, then centrifuge with ddH2O to collect the precipitated solid, which is the purified product BHET-MHET.

[0085] 2. Dissolve 10-20 mg of the purified product BHET-MHET in deuterated DMSO and collect one-dimensional NMR spectra using a 400 MHz NMR spectrometer.

[0086] BHET-MHET 1 The H-NMR spectrum is shown below. Figure 2 .

[0087] BHET-MHET 13 The C-NMR spectrum is shown below. Figure 3 .

[0088] The final structural formula of BHET-MHET is shown in equation (I):

[0089]

[0090] Following the steps described above, the purified enzyme of acyltransferase mutant A209T / S166V was replaced with acyltransferase mutants A209T, S166V, A209D, S166T, A209T / S166T, A209T / S166T / Q119R, or A209T / S166V / Q119I, while all other steps remained unchanged. The results showed that purified product BHET-MHET could be obtained in all cases.

[0091] Example 5: Quantitative analysis of the acyl transfer product BHET-MHET

[0092] 1. Prepare a 5mM BHET-MHET stock solution (acetonitrile as the solvent).

[0093] 2. Prepare BHET-MHET standards of different concentrations (1 mL for each standard) according to Table 2, filter them through a 0.22 μm filter membrane, and then perform high performance liquid chromatography (HPLC) analysis.

[0094] The HPLC analysis conditions were as follows: column: C18 reversed-phase column; temperature: 25℃; detection wavelength: 260nm; flow rate: 0.8mL / min; injection volume: 10μL; mobile phase: 0.1% formic acid, acetonitrile; elution conditions: 0-20min, gradient elution from 5% acetonitrile to 70% acetonitrile.

[0095] The results show that the standard curve for BHET-MHET is y = 0.1265x - 0.013, R0 2 =0.9994.

[0096] Table 2

[0097]

[0098] Example 6: Detection of acyltransferase activity of crude enzymes of acyltransferase mutants prepared in Example 2

[0099] 1. Preparation of the reaction system. The reaction system is 200 μL, consisting of 50 μL of pH 8.0, 100 mM phosphate buffer, 50 μL of the supernatant collected in Example 2 (i.e., the crude enzyme of PETase or the crude enzyme of the acyltransferase mutant) and 100 μL of diethyl terephthalate (BHET) solution with a final concentration of 15 mM (solvent: pH 8.0, 100 mM phosphate buffer).

[0100] 2. The yield of BHET-MHET in the reaction product was analyzed using high performance liquid chromatography (HPLC). The HPLC analysis conditions were the same as those in Example 5.

[0101] The test results are shown in Table 3. The results indicate that, compared with the crude PETase enzyme, the crude enzymes of most acyltransferase mutants showed a significantly higher maximum yield of BHET-MHET, approximately 1.2-16.7 times.

[0102] Table 3

[0103]

[0104]

[0105]

[0106] Example 7: Application of the crude enzyme of the optimal acyltransferase mutant prepared in Example 2 (i.e., acyltransferase mutant A209T / S166V) in the preparation of polyester prepolymers (e.g., products 1p, 2p, 3p, 4p, 5p, 6p, 7p, 8p, and 9p).

[0107] 1. Obtain standards for each substrate and each product. The structural formulas of each substrate and each product standard are detailed in Table 4.

[0108] Table 4

[0109]

[0110]

[0111] 10–20 mg of substrates 1–9, products 1p, 3p, 4p, 5p, 6p, 7p, 8p, and 9p were thoroughly dissolved in 500 μL of CDCl3, and one-dimensional NMR spectra were acquired using a 400 MHz NMR spectrometer. 10–20 mg of product 2p was thoroughly dissolved in 500 μL of deuterated DMSO, and one-dimensional NMR spectra were acquired using a 400 MHz NMR spectrometer.

[0112] Substrate 1 1 The H-NMR spectrum is shown below. Figure 4 .

[0113] Product 1p 1 The H-NMR spectrum is shown below. Figure 5 .

[0114] Substrate 2 1 The H-NMR spectrum is shown below. Figure 6 .

[0115] Product 2p 1 The H-NMR spectrum is shown below. Figure 7 .

[0116] Substrate 3 1 The H-NMR spectrum is shown below. Figure 8 .

[0117] Product 3p 1 The H-NMR spectrum is shown below. Figure 9 .

[0118] Substrate 4 1 The H-NMR spectrum is shown below. Figure 10 .

[0119] Product 4p 1 The H-NMR spectrum is shown below. Figure 11 .

[0120] Substrate 5 1 The H-NMR spectrum is shown below. Figure 12 .

[0121] Product 5p 1 The H-NMR spectrum is shown below. Figure 13 .

[0122] Substrate 6 1 The H-NMR spectrum is shown below. Figure 14 .

[0123] Product 6p 1 The H-NMR spectrum is shown below. Figure 15 .

[0124] Substrate 7 1 The H-NMR spectrum is shown below. Figure 16 .

[0125] Product 7p 1 The H-NMR spectrum is shown below. Figure 17 .

[0126] Substrate 8 1 The H-NMR spectrum is shown below. Figure 18 .

[0127] Product 8p 1 The H-NMR spectrum is shown below. Figure 19 .

[0128] Substrate 9 1 The H-NMR spectrum is shown below. Figure 20 .

[0129] Product 9p 1 The H-NMR spectrum is shown below. Figure 21 .

[0130] 2. Quantitative analysis of substrates 1-9 and products 1p-9p.

[0131] (1) Prepare mother liquors of substrate 1-substrate 9 and product 1p-product 9p at concentrations of 5mM, 10mM or 20mM (solvent is acetonitrile).

[0132] (2) The stock solutions of substrates 1-3 and products 1p-3p were diluted with 25% acetonitrile (the solvent consisted of 1 volume part acetonitrile and 3 volume parts pH 7.0, 20mM phosphate buffer) to prepare standard solutions of substrates 1, 2, 3, products 2p, and 3p at concentrations of 50, 100, 250, 500, 750, and 1000 μM, respectively. Standard solutions of product 1p at concentrations of 25, 50, 125, 250, 375, and 500 μM were also prepared. 1 mL of each standard solution was then filtered through a 0.22 μm filter and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis conditions were the same as those in Example 5.

[0133] The results show that the standard curve for substrate 1 is y = 0.0669x + 0.4967, R0 2 =0.9997; the standard curve for product 1p is y = 0.0961x - 1.3883, R 2 =0.9955; the standard curve for substrate 2 is y = 0.2422x - 0.2205, R 2 =1; the standard curve for product 2p is y = 0.443x - 9.5306, R 2 =0.9949; the standard curve for substrate 3 is y = 0.185x + 0.0209, R 2 =0.9999; the standard curve for product 3p is y = 0.4084x - 6.8961, R 2 =0.995.

[0134] (3) Dilute the mother solutions of substrate 4-substrate 9 and product 4p-product 9p with 80% acetonitrile (the solvent consists of 4 parts by volume of acetonitrile and 1 part by volume of pH 7.0, 20mM phosphate buffer) to prepare standard solutions of substrate 4, product 4p, substrate 7, product 7p, substrate 8 and product 8p with concentrations of 50, 100, 250, 500, 750 and 1000 μM, respectively. Prepare standard solutions of substrate 9 and product 9p with concentrations of 50, 100, 200, 250, 400 and 500 μM, respectively. Prepare standard solutions of substrate 5, substrate 6, product 5p and product 6p with concentrations of 1, 2, 3, 4 and 5 mM, respectively. Each standard solution was then taken in 1 mL, filtered through a 0.22 μm filter membrane, and analyzed by high performance liquid chromatography (HPLC). The HPLC analysis conditions were as follows: column: C18 reversed-phase column; temperature: room temperature; detection wavelength: 260 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: H2O, acetonitrile; elution conditions: 83% acetonitrile / 17% H2O.

[0135] The results show that the standard curve for substrate 4 is y = 0.0119x + 0.103, R02 =0.9999; the standard curve for product 4p is y = 0.0565x - 1.0896, R 2 =0.9976; the standard curve for substrate 5 is y = 16.7x + 0.6656, R 2 =0.9994; the standard curve for product 5p is y = 7.4979x - 0.6792, R 2 =0.9978; the standard curve for substrate 6 is y = 18.044x + 1.0662, R 2 =0.9949; the standard curve for product 6p is y = 4.1178x + 0.1525, R 2 =0.9998; the standard curve for substrate 7 is y = 0.0099x + 0.1301, R 2 =0.9996; the standard curve for product 7p is y = 0.0124x - 0.0425, R 2 =1; the standard curve for substrate 8 is y = 0.0071x + 0.0426, R 2 =0.9996; the standard curve for product 8p is y = 0.0121x - 0.2214, R 2 =0.9991; the standard curve for substrate 9 is y = 0.0042x + 0.0179, R 2 =0.9987; the standard curve for product 9p is y = 0.0075x - 0.1287, R 2 =0.983.

[0136] 3. Preparation of the reaction system. The reaction system consists of 2.5 mL of 1 mL of pH 6.5, 100 mM phosphate buffer, 1.5 mL of crude lyophilized enzyme powder of acyltransferase mutant A209T / S166V prepared in Example 2, and 0.1 g of substrate (BHET, substrate 1, substrate 2, substrate 3, substrate 4, substrate 5, substrate 6, substrate 7, substrate 8 or substrate 9).

[0137] 4. Take the reaction system prepared in step 3 and react at room temperature for 29 hours to obtain the reaction solution. Add 25% acetonitrile (the solvent consists of 1 volume part acetonitrile and 3 volumes of pH 7.0, 20mM phosphate buffer) to 20-30 μL of the reaction solution obtained with substrate BHET, substrate 1, substrate 2, or substrate 3 to dilute completely to the solid, with a dilution factor of 200-400 times. Add 80% acetonitrile (the solvent consists of 4 volumes acetonitrile and 1 volume of pH 7.0, 20mM phosphate buffer) to 20-30 μL of the reaction solution obtained with substrate 4, substrate 5, substrate 6, substrate 7, substrate 8, or substrate 9 to dilute completely to the solid, with a dilution factor of 50-300 times.

[0138] 5. The yields of the esterified products BHET-MHET, product 1p, product 2p, and product 3p corresponding to BHET, substrate 1, substrate 2, and substrate 3 were determined using high performance liquid chromatography (HPLC). The HPLC analysis conditions were the same as those in Example 5.

[0139] The yields of the esterified products 4p, 5p, 6p, 7p, 8p, and 9p corresponding to substrates 4, 5, 6, 7, 8, and 9 were determined by HPLC. The HPLC analytical conditions were as follows: column: C18 reversed-phase column; temperature: room temperature; detection wavelength: 260 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: H2O, acetonitrile; elution conditions: 83% acetonitrile / 17% H2O.

[0140] The test results are shown in Table 5. The results indicate that the acyltransferase mutant A209T / S166V can catalyze acyl transfer reactions of various substrates, and exhibits high catalytic efficiency for furan ring substrates (such as substrates 2 and 3). Among all tested substrates, substrate 3 showed the best conversion efficiency, with a product yield exceeding 90%. The catalytic efficiency for benzoate substrates (such as substrates 7, 8, and 9) was lower, below 10%.

[0141] Table 5

[0142]

[0143]

[0144] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, characterized by: It is a protein obtained by only the following mutations based on the amino acid sequence of the hydrolase PETase shown in SEQ ID NO: 1: A209T, A209V, A209T / S166V, A209T / S166V / Q119I, A209T / S166T / Q119I, A209T / S166T, A209T / S166T / Q119R, A209T / Q119R, A209N, A209G, A209I, A209C, A209F, A209Q, A209R, A209M, A209D, A209P, A209L, A209K, A209H, A209Y, or A209W.

2. A nucleic acid molecule encoding the protein of claim 1.

3. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule of claim 2.

4. The use of the protein of claim 1, the nucleic acid molecule of claim 2, or an expression cassette containing the nucleic acid molecule of claim 3, a recombinant vector, or a recombinant microorganism in the preparation of acyltransferases.

5. The application of the protein of claim 1, the nucleic acid molecule of claim 2, or an expression cassette containing the nucleic acid molecule of claim 3, a recombinant vector, or a recombinant microorganism in catalyzing the acyl transfer reaction of BHET to obtain BHET-MHET: in, The structural formula of BHET-MHET is as follows: 。 6. The application of acyltransferase mutants, their encoded nucleic acid molecules, or expression cassettes containing said nucleic acid molecules, recombinant vectors, or recombinant microorganisms in catalyzing acyl transfer reactions of aromatic, furan, or aliphatic polymer precursors; wherein the polymer precursors are selected from: ； The products obtained from the reaction are: ； The acyltransferase mutant described therein is a protein obtained by the A209T / S166V mutation based on the amino acid sequence of the hydrolase PETase shown in SEQ ID NO: 1.

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